Deferoxamine Treatment Combined With Sevoflurane Postconditioning Attenuates Myocardial Ischemia-Reperfusion Injury by Restoring HIF-1/BNIP3-Mediated Mitochondrial Autophagy in GK Rats

Figure 1. Schematic diagram of ischemia reperfusion in Sprague-Dawley (SD) rats. Except the Sham group, all hearts were subjected to 40 min ischemia, followed by reperfusion for 120 min. The SpostC was executed by administration of 2.4% sevoflurane (1.0 MAC) for 15 min at onset of reperfusion, and then reperfusion for 105 min. MSP and 2ME2 groups were intraperitoneally injected with 2ME2 (15 mg/kg) before ligation of LAD, and the remaining processing is the same as SpostC or I/R groups.

Figure 2. Schematic diagram of ischemia reperfusion in Goto-Kakizaki (GK) rats. Except the Sham group, all hearts were subjected to 40 min ischemia, followed by reperfusion for 120 min. DFO (200 mg/kg) was given intraperitoneally 24 h before the experiment. SpostC and 2ME2 are treated as above ( Figure 1 ).

Figure 3. SPostC promoted mitochondrial autophagy by up-regulating HIF-1/BNIP3 signaling pathway protein expression in non-diabetic state. SpostC increased the expression levels of HIF-1α, BNIP3, and LAMP2, while decreased the expression levels of LC3BII, p62 and Beclin-1. (A) Expression of HIF-1α. (B) Expression of BNIP3. (C) Expression of Beclin-1. (D) Expression of LC3BII. (E) Expression of p62. (F) Expression of LAMP2. (^P < 0.05 vs I/R, #P < 0.05 vs SPostC) (n = 5/group).

Figure 4. SPostC reduced the damage of mitochondrial ultrastructure and the accumulation of autophagosomes in non-diabetic state. (A) Representative transmission electron micrographs show mitochondrial ultrastructure and autophagosomes in the ischemic myocardial after 2 h of reperfusion. (B) Quantitative analysis of autophagosome. Autophagosome are indicated by the black arrow. The transmission electron microscope image was magnified 20,000 times. (^P < 0.05 vs I/R, #P < 0.05 vs SPostC) n = 5/group).

Figure 5. SPostC attenuated myocardial mitochondrial function damage after ischemia-reperfusion in non-diabetic state. SpostC increased ATP content, stabilized membrane potential, and reduced ROS production. (A) ATP content. (n = 6/group) (B) Mitochondrial membrane potential. (n = 6/group) (C) Quantitative analysis of DHE-positive nuclei. (D) ROS Fluorescent Probe-DHE. ROS were stained with DHE (red) and nucleus with DAPI (blue). (n = 6/group) (^P < 0.05 vs I/R, #P < 0.05 vs SPostC, Scale bars 50 um).

Figure 6. SPostC reduced myocardial infarct size in non-diabetic state. White color represents myocardia infarct area, red color represents the area at risk. The myocardial infarct size was calculated for each slice, and reported as the percent of infarct divided by the total area at risk. (^P < 0.05 vs I/R, #P < 0.05vs SPostC) (n = 6/group).

Figure 7. SPostC improved cardiac function after ischemia-reperfusion in non-diabetic state. (A) Representative M-mode images of echocardiography. (B) FS, fractional shortening. (C) EF, ejection fraction. (^P < 0.05 vs I/R, #P < 0.05 vs SPostC) (n = 6/group).

Figure 8. After deferoxamine (DFO) treatment, SPostC promoted mitochondrial autophagy by up-regulating HIF-1/BNIP3 signaling pathway protein expression in diabetic state. SpostC increased the expression levels of HIF-1α, BNIP3 and LAMP2, while decreased the expression levels of LC3BII, p62, and Beclin-1. (A) Expression of HIF-1α. (B) Expression of BNIP3. (C) Expression of Beclin-1. (D) Expression of LC3BII. (E) Expression of p62. (F) Expression of LAMP2. (^P < 0.05 vs I/R, #P < 0.05 vs DFO, &P < 0.05 vs DFO+SpostC) (n = 5/group).

Figure 9. After deferoxamine (DFO) treatment, SPostC reduced the damage of mitochondrial ultrastructure and the accumulation of autophagosomes in diabetic state. (A) Representative transmission electron micrographs show mitochondrial ultrastructure and autophagosomes in the ischemic myocardial after 2 h of reperfusion. (B) Quantitative analysis of autophagosome. Autophagosome are indicated by the black arrow. The transmission electron microscope image was magnified 20,000 times. (^P < 0.05 vs I/R, #P < 0.05 vs DFO, &P < 0.05 vs DFO+SpostC) (n = 5/group).

Figure 10. After deferoxamine DFO treatment, SPostC attenuated myocardial mitochondrial function damage after ischemia-reperfusion in diabetic state. SpostC increased ATP content, stabilized membrane potential, and reduced ROS production after DFO treatment. (A) ATP content. (n = 6/group) (B) Mitochondrial membrane potential. (n = 6/group) (C) Quantitative analysis of DHE-positive nuclei. (D) ROS Fluorescent Probe-DHE. ROS were stained with DHE (red) and nucleus with DAPI (blue). (n = 6/group) (^P < 0.05 vs I/R, #P < 0.05 vs DFO, &P < 0.05 vs DFO+SpostC, Scale bars 50um).

Figure 11. After deferoxamine (DFO) treatment, SPostC reduced myocardial infarct size in diabetic state. White color represents myocardia infarct area, red color represents the area at risk. The myocardial infarct size was calculated for each slice, and reported as the percent of infarct divided by the total area at risk. (^P < 0.05 vs I/R, #P < 0.05 vs DFO, &P < 0.05 vs DFO+SpostC) (n = 6/group).

Figure 12. After deferoxamine (DFO) treatment, SPostC improved cardiac function after ischemia-reperfusion in diabetic state. (A) Representative M-mode images of echocardiography. (B) FS, fractional shortening. (C) EF, ejection fraction. (^P < 0.05 vs I/R, #P < 0.05 vs DFO, &P < 0.05 vs DFO+SpostC) (n = 6/group).